Project Summary The dynamic microtubule cytoskeleton mediates intracellular organization, generates forces in dividing or migrating eukaryotic cells, and forms tracks for intracellular trafficking. The fundamental properties of microtubules, including polarized growth and ?dynamic instability?, stem directly from the activities of the microtubule building blocks, the ?- and ?-tubulin heterodimers. Three conserved tubulin cofactors and dedicated Arf-like 2 G-protein form multi-subunit platforms for the biogenesis and degradation of ??-tubulin dimer, leading to a high concentration within the cytoplasm. The mechanisms for these assemblies remain mostly mysterious, due in part to a lack of structural information. In addition, we do not understand how conserved microtubule polymerases with arrays of Tumor Overexpressed Gene (TOG) domains recruit ??-tubulins and accelerate their incorporation while tracking dynamic microtubule ends. Understanding these cellular pathways is critical since genetic defects that impair either soluble ??-tubulin biogenesis or microtubule polymerases are linked to inherited neurological and developmental disorders and are observed in human cancers, respectively. This proposal explores the biochemical and physical mechanisms of ??-tubulin biogenesis and microtubule polymerase assemblies and their impact on microtubule function. Our strategy combines methods across multiple resolution scales, including in vitro reconstitution of purified protein assemblies, structural studies by cryo-electron microscopy (cryo- EM), reconstitution of assemblies with microtubule dynamics using in vitro fluorescence microscopy-based assays, and in vivo live imaging with microtubules within living cells. First, we will determine structural transitions describing ??-tubulin biogenesis assemblies and their functional impact of ??-tubulin biogenesis and degradation. During the previous period, we established reconstitution system for these assemblies with ??-tubulin and describe cryo-EM structural studies leading to medium resolution structures in complex with ??-tubulins. 1) We will determine structural states for the ??-tubulin biogenesis assemblies in multiple biochemical states using high-resolution cryo-EM to understand how these assemblies catalyze dimerization of ??-tubulin and its degradation. 2) We will dissect functional roles of structural elements and interactions within current structures to determine their role in the ??-tubulin biogenesis process using in vitro and in vivo methods. Second, we will examine the mechanisms of microtubule polymerases with arrays of TOG domains their regulatory mechanisms. In the previous period, we describe a new model for ??-tubulin recruitment and polymerization by TOG domain arrays as microtubule polymerases, developed based on our structural and biochemical studies. We validated this model using in vitro reconstitution and in vivo live imaging of structure-based designer defective mutants, revealing that the ??-tubulin accelerating and processive plus-end tracking activities originate from unique features in TOG domain arrays. 1) We will study mechanisms of super-complexes of microtubule polymerase in complex with their activators, the transforming acidic coiled-coil proteins, in by using well- explored structural, in vitro reconstitution and in vivo live imaging strategies. 2) Determine the structural and functional relevance of our new model to mammalian microtubule polymerases with their unique pentameric TOG domain array arrangement using cryo-EM structural studies, in vitro reconstitution of designer mutants, and in vivo imaging approaches of structure-based mutants. We expect these studies to yield new structural and biophysical data, which will refine our new models will deepen our understanding of soluble ??-tubulin biogenesis, recruitment and incorporation during microtubule polymerization. This understanding will in turn point toward new strategies for addressing defects in tubulin biogenesis and regulation, potentially impacting patients with a range of developmental and neurological disorders.